2. Playing around with media conditions

Authors

Learning outcomes

In this tutorial you will use cobrapy to learn how to:

Setup

2.1 Growth medium

The availability of nutrients has a major impact on metabolic fluxes and cobrapy provides some helpers to manage the exchanges between the external environment and your metabolic model. In experimental settings the “environment” is usually constituted by the growth medium, i.e. the concentrations of all metabolites and co-factors available to the modeled organism. However, constraint-based metabolic models only consider fluxes. Thus, you cannot simply use concentrations since fluxes have the unit mmol / [gram of dry-cell weight * hour] (i.e. concentration per gram dry weight of cells and hour).

Also, you are setting an upper bound for the particular import flux and not the flux itself. There are some crude approximations. For instance, if you supply 1 mmol of glucose every 24h to 1 gram of bacteria you might set the upper exchange flux for glucose to 1 mmol / [1 gDW * h] since that is the nominal maximum that can be imported. There is no guarantee however that glucose will be consumed with that flux. Thus, the preferred data for exchange fluxes are direct flux measurements as the ones obtained from timecourse exa-metabolome measurements for instance.

So how does that look in COBRApy? The current growth medium of a model is managed by the medium attribute.

This will return a dictionary that contains the upper flux bounds for all active exchange fluxes (the ones having non-zero flux bounds). Right now we see that we have enabled aerobic growth. Let's optimize to check the growth in the given medium.

You can modify a growth medium of a model by assigning a dictionary to model.medium that maps exchange reactions to their respective upper import bounds. Let's edit the medium to have ethanol as carbon source instead of glucose.

As we can see, the glucose in our medium has been replaced by ethanol. Let's check how it influences the growth rate.

The growth rate is reduced by around one order of magnitude when growing on ethanol compared to glucose. In conclusion, we have the same metabolic reconstruction (iMM904), simulated under two different media conditions using different carbon sources. model_yeast is growing with glucose as a carbon source while model_yeastv2 is using ethanol.

Questions

  1. Can the yeast model grow using lactose as a carbon source? What about galactose? Was this an expected result?
  2. What happens when you lower the uptake rate of ammonium? How about when it is set to 0? Was this expected?
  3. Can you quantify the changes in growth rate induced by replacing ammonium with an amino acid of your choice to the media? e.g. EX_val__L_e

Tip #1

Tip #2

Answers

Question 1

Yeast cannot grow on lactose, as it cannot break it down into simple sugars [ref]. The model reflects this fact, as it is missing an exchange reaction for lactose. When we try to modify the uptake rate of lactose we get an error, because the model does not have an exchange reaction for lactose. On the other hand, yeast can indeed grow on galactose.

Question 2

Lowering the ammonium uptake rate causes a decrease in growth rate. Setting the uptake rate to 0 results in near-zero growth rate. This is expected, as yeast needs a nitrogen source in order to grow [ref].

Question 3

Replacing ammonium with valine results in ~2.2% increase in growth rate.

2.2 Gene essentiality

A gene is considered essential if restricting the flux of all reactions that depend on it to zero causes the objective, e.g., the growth rate, to also be zero, fall below a given threshold, or result in infeasible solutions.

Now we will use the function find_essential_genes to find essential genes in both conditions, extract the IDs of the genes, and compare the two lists. Can you do it on your own?

First lets check the model growing with ethanol as a carbon source:

Next lets check the model growing with glucose as a carbon source:

Check how large the intersect is between the two lists:

Lets check which genes are unique to each condition:

Questions

  1. Why there are more essential genes for growth on ethanol than for growth on glucose?
  2. What pathways are these ethanol-growth essential genes associated with?
  3. Choose an essential gene unique to growth in ethanol and search the literature for its function. Does it make sense to you that it is essential?

Tip

For an overview of central carbon metabolism in yeast refer to the figure below, obtained from this publication.

image.png

Answers

Question 1

Because growing on ethanol requires an extra set of enzymes which are not needed for growth on glucose. Growing on glucose requires enzymes for glycolysis only, while growing on ethanol additionally requires aerobic fermentation enzymes.

Question 2

These genes are associated with the TCA cycle and oxidative phosphorylation (i.e. electron transport chain).

Question 3

A search for the gene YLR395C reveals that it is indeed associated with the electron transport chain and cellular respiration.

2.3 Simulating the Crabtree effect

Yeast central carbon metabolic pathways: all short-term Crabtree positive yeasts possess an upregulated aerobic (blue) and anaerobic (red) glycolytic pathway, even under fully aerobic conditions, when energy and carbon-source is limiting. At low glucose uptake rates, yeasts cells are purely respiring and there is a carbon-flux only through glycolysis (GF) and respiration (RF). Upon a sudden glucose excess condition (glucose pulse), the glycolytic flux will exceed the respiratory flux, which results in a fermentative flux (FF) and ethanol production. In other words, it appears as if slowly dividing short-term Crabtree positive cells, with little food around are already equipped with a strong energy producing apparatus, for rapid glucose consumption and energy production.

image-3.png (Adapted from Hagman and Piškur, 2015)

To simulate the Crabtree effect, we consider a fixed amount of glucose and different concentrations of oxygen in our medium.

Plot results

Questions

  1. Can you explain the observed behavior in the plot above?
  2. Try playing around with other media components, for example, what happens if you keep oxygen fixed and vary glucose?
  3. Literature search question: What are two similarities and two differences between the Crabtree and Warburg effects?

Tip

Check out this publication describing the Crabtree and Warburg effects. In particular, have a look at Figure 1 shown below:

image-2.png

Answers

Question 1

The plot shows the Crabtree effect. When glucose is present in excess and under anaerobic/low oxygen conditions, we see that ethanol production reaches a maximum. Furthermore, we see that as oxygen uptake increases, yeast reduces its fermentative flux but it does not reach zero.

Question 2

One of the conditions of the Crabtree effect is that the carbon source (i.e. glucose) has to be present in excess. In line with this precondition, we see that very little ethanol is produced (i.e. low fermentative flux) until glucose is no longer limiting.

Question 3

Similarities:

Differences: